Early disruption of centromeric organization in A (Cenpa) null mice

Emily V. Howman, Kerry J. Fowler, Ainsley J. Newson, Saara Redward, Andrew C. MacDonald, Paul Kalitsis, and K. H. Andy Choo*

The Murdoch Institute, Royal Children’s Hospital, Flemington Road, Parkville 3052, Australia

Edited by John A. Carbon, University of California, Santa Barbara, CA, and approved November 23, 1999 (received for review October 5, 1999) Centromere protein A (Cenpa for mouse, CENP-A for other species) reveals embryonic lethality at 2.5 days pc, accompanied by is a H3-like protein that is thought to be involved in the enlarged nuclei containing an increased number of nucleoli, nucleosomal packaging of centromeric DNA. Using targeting, nuclear bridging, condensation, and spindle fiber we have disrupted the mouse Cenpa gene and demonstrated that bundling (21). the gene is essential. Heterozygous mice are healthy and fertile The role of CENP-A in centromeric function has yet to be whereas null mutants fail to survive beyond 6.5 days postconcep- elucidated. CENP-A is a histone-H3 like protein that is con- tion. Affected embryos show severe mitotic problems, including served in mammals (22, 23) and S. cerevisiae (24). The C terminal micronuclei and macronuclei formation, nuclear bridging and bleb- (residues 48–135) of CENP-A is 62% identical to that of histone bing, and chromatin fragmentation and hypercondensation. Im- H3 and corresponds to the histone fold domain. The histone fold munofluorescence analysis of interphase cells at day 5.5 reveals domain consists of three ␣-helices (HI, HII, and HIII) separated complete Cenpa depletion, diffuse Cenpb foci, absence of discrete by two ␤-sheet structures (strand A and strand B) (25) (see Fig. Cenpc signal on , and dispersion of Cenpb and Cenpc 1). This domain of has been shown to be sufficient throughout the nucleus. These results suggest that Cenpa is es- for assembly in vitro (26) and in vivo (27). There is sential for targeting of Cenpc and plays an early role no similarity seen between the N-termini sequences (residues in organizing centromeric chromatin at interphase. The evidence is 1–47) of CENP-A and normal histone H3 (2). Although this consistent with the proposal of a critical epigenetic function for divergence initially was thought to provide CENP-A with the CENP-A in marking a chromosomal region for centromere forma- centromere targeting property, a histone H3 chimeric protein tion. containing the N terminus of CENP-A and the histone H3 histone fold domain failed to localize to the centromere, indi- kinetochore ͉ epigenetic ͉ gene targeting cating that the C-terminal end is responsible for centromere targeting (28). CENP-A synthesis appears to be coupled with he centromere is an essential chromosomal component centromere replication during mid-S to early , whereas Trequired for the faithful segregation of during histone H3 expression peaks early in S phase (28). Expression of and . The kinetochore is a DNA-protein complex CENP-A under the histone H3 promoter fails to localize at the comprising both constitutive that are present at the centromere (28). These studies suggest that CENP-A is involved centromere throughout the and transient proteins that in the packaging of centromeric chromatin and that the protein are present at various stages (1). Three of the best-studied may provide an early epigenetic marker for centromere forma- constitutive proteins are centromere proteins CENP-A, tion (29). CENP-B, and CENP-C. CENP-A is a 17-kDa histone H3-like Only limited functional data are available for CENP-A. Mi- croinjection of antibodies raised against the N terminus of protein involved in centromeric nucleosome formation (ref. 2; ͞ described below). CENP-B is an 80-kDa protein that binds a CENP-A into HeLa cells within 3 hr of G1 S release resulted in 17-bp motif known as the CENP-B box, which is present in interphase arrest (30). Highly condensed nuclei, granular cyto- human ␣-satellite and mouse minor satellite DNA (3, 4). Gene plasm, and loss of capability were observed. Anti- knockout analysis of Cenpb in mice indicates that this protein is body injection in midinterphase did not disrupt mitosis; however, not essential (5–7), although a decrease in body weight and testis a mitotic lag was observed possibly because of the antibody size accompanied protein deficiency (5). CENP-C is a 140-kDa interfering with attachment (31). Studies on CSE4p protein that interacts with chromatin at the inner kinetochore ( protein), an S. cerevisiae homolog of plate (8). In vitro DNA binding studies suggest that CENP-C may CENP-A, have demonstrated the protein to be a component of bind to DNA (9). CENP-C null mutation results in embryonic the core centromere (32). Mutation in CSE4p results in misseg- lethality at 3.5 days postconception (pc), with a missegregation regation and cell arrest in mitosis; however, the increase in phenotype and metaphase arrest (10, 11). Metaphase arrest also chromosome loss is slight (33). The arrest phenotype is consis- is observed after microinjection of anti-CENP-C antibodies at tent with a specific cell division block that appears to occur after interphase (12). CENP-C shares a region of homology with Mif2, the mitotic spindle has formed but before the onset of anaphase a Saccharomyces cerevisiae protein. Mutations in the MIF2 gene (33). The arrested cells have a 2n DNA content, indicating that result in defective chromosome segregation and delayed pro- DNA replication has taken place before arrest. gression through mitosis (13). However, CENP-C alone is not

sufficient to induce centromeric formation (14). This paper was submitted directly (Track II) to the PNAS office. A number of transient centromere proteins now have been Abbreviations: ES, embryonic stem; CENP, centromere protein; INCENP, inner CENP; pc, described (1, 15–19). Of particular relevance to the present study postconception; IRES, internal ribosome-entry site. involving the use of the gene targeting technique is the inner *To whom reprint requests should be addressed. E-mail: [email protected]. CENP (INCENP). This protein localizes to the centromere at The publication costs of this article were defrayed in part by page charge payment. This early mitosis and is present on the metaphase plate at the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. metaphase-anaphase transition (20). Gene disruption in mice §1734 solely to indicate this fact.

1148–1153 ͉ PNAS ͉ February 1, 2000 ͉ vol. 97 ͉ no. 3 Downloaded by guest on September 29, 2021 Fig. 2. Southern blotting and PCR genotyping of cell line, tail, and embryo DNA. (a) Southern blot analysis of putative targeted ES cell colonies after EcoRI digestion and probed with an external probe (see Fig. 1 b and d). (b) PCR analysis of mouse tail DNA showing a wild-type product of 455 bp detected by WT-1 and WT-2 primers, and a targeted product of 750 bp detected by N-1 and Fig. 1. Targeted disruption of the mouse Cenpa gene. (a) The mouse Cenpa WT-2 primers. SA, splice-acceptor site. (c) Nested PCR of mouse embryos protein showing the different subdomains, in particular those at the C termi- resulting in a 135-bp wild-type product when using primers MA1, MA2, MA3, nus that are required for centromere targeting. Our targeting construct (see and MA4, and a 248-bp targeted product when using primers GF1, GR1, GF2, below) was designed to delete amino acids 29–64 (gray box), which will and GR2. effectively remove the entire centromere-targeting domain. (b) A restriction map of the Cenpa gene. The exons are denoted by black boxes (23).(c) The gene replacement construct, where the selectable marker cassette consists of by Southern blot (Fig. 2a). Chimeric mice were produced as a splice-acceptor site (SA), a picornaviral IRES, a lacZ-neomycin-resistance fusion gene, and a simian virus 40 polyadenylation sequence (PA). (d) The described (21). Mice were genotyped by PCR. DNA was ex- Cenpa after gene disruption. The positions of external probes used in tracted from mouse tails as described (21). Primers designed by Southern analysis are shown and the expected size fragments are 7.9-kb using GENEWORKS were: WT-1 (5Ј-TCAGACACTGCGCA- wild-type allele and a 4.8-kb targeted allele. ATG and TAA are translation start GAAGAC); WT-2 (5Ј-GAGCTTAGGAACTGGCATGG); and stop codons, respectively. Restriction enzymes used were SacI (S), SalI (Sa), and N-1 (5Ј-TTCTATCGCCTTCTTGACGAG) (Fig. 2b). EcoRI (E), XbaI (Xb), XhoI (Xh), KpnI (K), NheI (Nh), and SpeI (Sp). Genotyping of Preimplantation Embryos. Preimplantation embryos To further understand the role of CENP-A in centromere were obtained from heterozygous mice. Breeding pairs were function, we have used the technique of gene targeting by examined daily for vaginal plugs (an indicator of 0.5 days homologous recombination to enable the production of Cenpa gestation) and denoted as 0.5 days pc. A nested PCR protocol null mice. Our analysis of Cenpa null mutants has enabled us to was designed and used for the amplification of the 2.5-day elucidate the involvement of this protein in mitotic cell division embryonic DNA. Embryos were flushed and transferred to PCR ␮ and, in particular, its role in kinetochore assembly. tubes in 25 lofdH2O. Nest 1a: denaturation at 95°C for 15 min. ϫ Nest 1b: addition of 10 buffer (containing 15 mM MgCl2) Methods (Perkin–Elmer), 0.2 mM dNTP, 250 ng of wild-type primers Construction of Targeting Vectors. The targeting construct contains MA1, MA2, and LacZ-neomycin primers GF1 and GR1, 1 unit 6.4 kb of the Cenpa gene (23) and was used to delete exon 2 of AmpliTaq DNA Polymerase (Perkin–Elmer) in a final volume ␮ (amino acids 29–64) and disrupt the protein through the intro- of 50 l. Cycle 1: 95°C for 2 min; 55°C for 3 min; 72°C for 90 sec. duction of the selectable marker cassette. Exon 2 was deleted via Cycles 2–30: 95°C for 60 sec; 57°C for 60 sec, and 72°C for 90 sec. the flanking NheI and XbaI sites followed by the insertion of a Nest 2: Using 1 ␮l of the Nest 1b product, add 10ϫ buffer, 0.2 selectable marker cassette isolated from pGT1.8IRES.␤geo, mM dNTP, primers MA3, MA4, GR2, GF2, 1 unit of AmpliTaq, where IRES is the internal ribosome-entry site (34). This in a final volume of 25 ␮l. Cycle 1: 95°C, 2 min; 58°C, 60 sec; 72°C, construct, when homologously recombined into the mouse 90 sec. Cycles 2–30: 95°C, 1 min; 58°C, 1 min; 72°C, 90 sec. Cenpa locus, will result in a truncated protein lacking the Oligonucleotide primer sequences were: MA1 (5Ј-TGGAACT- centromere targeting domain (Fig. 1). GCAGTCTGGGAAC); MA2 (5Ј-TCTGTCTTCTGCGCAGT- GTC); GF1 (5Ј-AGTATCGGCGGAATTCCAG); GR1 (5Ј-G- Generation of Targeted Embryonic Stem (ES) Cells and Mice. Mouse ATGTTTCGCTTGGTGGTC); MA3 (5Ј-CCCAAAGCTCA- ES cells (129͞1) were electroporated with 40 ␮g of linearized GAGCAAATTC); MA4 (5Ј-AGTATGTGGCAGCACAG- construct DNA, grown on STO͞NeoR feeders, and growth- CAG); GR2 (5Ј-CCTCGTCCTGCAGTTCATGTCTGGTG); selected by using G418 (21). Resistant colonies were genotyped and GF2 (5Ј-CCATTACCAGTTGGTCTGGTG) (Fig. 2c).

Howman et al. PNAS ͉ February 1, 2000 ͉ vol. 97 ͉ no. 3 ͉ 1149 Downloaded by guest on September 29, 2021 Genotyping of Day-8.5 Embryos. Individual embryos were dissected Table 1. PCR genotyping of embryos at days 2.5 and 8.5 pc, from their implantation site at 8.5 days gestation, washed twice showing the number of embryos and, in brackets, % of total in PBS, and transferred to a microfuge tube. Mouse tail lysis Genotype Day 2.5 Day 8.5 buffer and proteinase K (1 ␮g͞ml) were added and incubated at 50°C for 4 hr. DNA was extracted twice with phenol-chloroform ϩ͞ϩ 3 (14%) 11 (30%) and once with chloroform followed by ethanol precipitation ϩ͞Ϫ 7 (34%) 18 (49%) using 1 ␮l of glycogen. This precipitate was resuspended in 20 ␮l Ϫ͞Ϫ 4 (19%) 0 of Tris-EDTA from which 5 ␮l was used for each PCR. No PCR result 7 (33%) 0 Abortive implantation sites N.R. 8 (21%) Culturing of Mouse Embryos. A culturing protocol was introduced Total no. of embryos 21 37 to assess embryo development between 3.5 and 8.5 days pc. N.R., not relevant because embryos have not implanted at day 2.5. Embryos were flushed at 3.5 days and cultured in 15-mm diameter dishes (Nunc) with ES media supplemented with leukemia inhibiting factor (AMRAD, Melbourne, Australia) at heterozygous targeted cell lines into blastocysts resulted in four 37°C, 5% CO2. Embryos were photographed daily and harvested germ-line chimeras. These mice were crossed with C57BL͞6to at 8.5 days if normal in appearance or earlier if signs of produce heterozygous mice (Fig. 2b). degeneration were apparent. The embryos were rinsed in PBS and treated with 0.25% trypsin for 3–5 min, resulting in detach- Embryonic Lethality of Cenpa Null Offspring Occurs Postimplantation ment of the trophectoderm cells. Micro-glass pipettes were used Between Days 3.5 and 8.5 pc. The heterozygous mice were phe- to collect the cells. The PCR protocol was the same as that used notypically normal with no obvious impairment of growth or for the 8.5-day embryos (see Fig. 2c). fertility. Intercrossing of heterozygous mice resulted in a total of 186 progeny of which 63 (34%) were ϩ͞ϩ and 123 (66%) were Giemsa Staining of Embryos. Embryos (2.5 and 3.5 days) were ϩ͞Ϫ, indicating embryonic lethality of the homozygous mutant placed in M16 media (Sigma) under oil, then transferred to a state. Embryonic lethality was also evident from the reduced microwell containing 0.6% trisodium citrate for 4–8 min. Indi- Ϯ ϩ͞Ϫϫϩ͞Ϫ ϭ vidual embryos were placed on glass slides and fixed in a droplet average litter size of 6.0 2.4 for crosses (n 23 ͞ litters), compared with 9.1 Ϯ 2.6 and 8.6 Ϯ 3.3 for the ϩ͞ϩϫ of methanol acetic acid (3:1). After two rinses in fixative, the ϩ͞ϩ ϭ ϩ͞Ϫϫϩ͞ϩ ϭ embryos were stained with 10% Giemsa in PBS, pH 6.8 (Gurr), (n 31 litters) and (n 13 litters) crosses, for 10 min, air-dried, and mounted in DPX (BDH) for analysis. respectively. To enable morphological analysis of 4.5-, 5.5-, and 6.5-day To determine the point of Cenpa null lethality, embryos at 2.5 embryos, embryos were cultured on gelatinized (0.1% gelatin in and 8.5 days pc were genotyped by nested PCR (Fig. 2c). Matings ϩ͞Ϫϫϩ͞Ϫ PBS) coverslips (22 mm ϫ 22 mm) in 35-mm Petri dishes (Nunc). of yielded 21 embryos at 2.5 days and 37 embryos Embryos that failed to attach to the coverslips were harvested at 8.5 days. Genotyping by PCR indicated that null mutants were and treated in the same manner as the 2.5- and 3.5-day embryos. viable (and healthy looking by Giemsa staining; not shown) at 2.5 Embryos were fixed in methanol͞acetic acid for 10 min, stained days (Table 1). The results indicated that the time of embryo as above, and examined on an Olympus 1ϫ70 micro- death apparently had occurred before 8.5 days because no scope͞Nikon F-601 camera system. homozygous mutants were detected. Examination of the uteri of female mice for the 8.5-day embryos revealed eight (21%; Table Mitotic Index Determination. Cultured embryos were either 1) abortive implantation sites. These abortive sites were either Giemsa-stained (as above) or fixed in 2% paraformaldehyde and empty or contained remnants of highly degenerate and resorbing mounted in Vectorshield containing 4Ј,6-diamidino-2- embryos. This analysis indicated that the point of embryonic phenylindole (DAPI). DAPI staining of embryos was used to lethality occurred postimplantation between 3.5 and 8.5 days. enable mitotic detection in various focal planes. The number of cells undergoing mitosis was calculated as a percentage of the Morphological Degeneration of Cultured Cenpa Null Embryos at Day total cell number using both methods. 5.5 pc. To further investigate postimplantation development, embryos from ϩ͞Ϫϫϩ͞Ϫ crosses were flushed at 3.5 days and Immunofluorescence Analysis of Embryos. Embryos were cultured cultured individually. The embryos were photographed daily by on coverslips, rinsed twice in PBS, fixed for 5 min in 2% phase contrast or stained with Giemsa, and the images were paraformaldehyde in PBS, permeabilized with 0.1% Triton captured for more detailed examination. (In the following X-100 in PBS for 2 min, and rinsed an additional two times in discussion, the age of cultured embryo continues to refer to the PBS (35). Antibodies were applied to coverslips for 1 hr at 37°C. number of days pc; that is, 3.5 days in utero plus days in culture.) The coverslips were washed three times for 5 min in 1ϫ KB At 5.5 days pc (i.e., 2 days in culture), the inner cell mass of a buffer (10 mM Tris⅐HCl͞15 mM NaCl͞0.1% BSA), the second- ϩ͞ϩ or ϩ͞Ϫ embryo was prominent and surrounded by ary antibody was applied for 1 hr and washing was repeated. The coverslips then were rinsed in PBS and stored in PBS at 4°C. trophectoderm outgrowth after attachment to the coverslip (Fig. Embryos were mounted in 4Ј,6-diamidino-2-phenylindole. Im- 3 a and b) (the inner cell mass subsequently would give rise to age analysis was performed by using an Axioskop fluorescence the embryo proper whereas the trophectoderm would form the microscope equipped with a 63ϫ objective (Zeiss), a charge- extra-embryonic tissue). In a number of embryos, degeneration coupled device camera (Photometrics Image Point, Tucson, AZ) of the inner cell mass and trophectoderm cells was apparent (Fig. and IPLAB software (Signal Analytics, Vienna, VA). 3 d and e). Rapid degeneration of these embryos continued and, at 6.5 days, a defined inner cell mass was no longer visible, while Results the trophectoderm cell number also declined dramatically (Fig. Generation of Cenpa Heterozygous Cell Lines and Mice. The Cenpa- 3f). The remaining embryos maintained a healthy inner cell neoR construct (Fig. 1c) was transfected into 129͞1 ES cells mass, trophectoderm growth, and morphology from day 6.5 (Fig. grown on STO͞NeoR feeder cells and placed under G418 3c) to day 8.5. Healthy embryos were harvested at day 8.5 selection for 7–10 days. Screening of 48 resistant clones by whereas the degenerating embryos were harvested on day 6.5 for Southern blot analysis gave 26 positive clones (Fig. 2a), indicat- PCR genotyping. The results indicated that all of the day-8.5 ing a targeting efficiency of 54%. Microinjection of these embryos were either ϩ͞ϩ or ϩ͞Ϫ, whereas all of the day-6.5

1150 ͉ www.pnas.org Howman et al. Downloaded by guest on September 29, 2021 Fig. 3. Phase contrast and Giemsa staining images of day-5.5 and -6.5 embryos. Day-5.5 normal embryo photographed by phase (a) or stained with Giemsa (b). (c) Day-6.5 normal embryo stained with Giemsa. Note the compact, dark inner cell mass and the surrounding trophectoderm outgrowth. Day-5.5 Ϫ͞Ϫ embryo photographed by phase (d) or stained with Giemsa (e). (f) Day-6.5 Ϫ͞Ϫ embryo stained with Giemsa. Note the absence of a defined inner cell mass and the incoherent cells in both the day-5.5 and -6.5 Ϫ͞Ϫ embryos. Magnification for a–f: ϫ150. (g and h) Close-ups of e and (i and j) close-ups of f, showing micronuclei (empty-triangle arrow), macronuclei (filled-triangle arrow), nuclear bridging (open arrow), nuclear blebbing (filled arrowhead), and highly condensed chromatin bodies (empty arrowhead). GENETICS

degenerating embryos showed a 100% coincidence with the 6.5 days, most of the cells were macronucleated, suggesting that Ϫ͞Ϫ genotype (Table 2). chromosome segregation and normal cytokinesis had come to a halt. When the mitotic indices of day-5.5 cultured embryos were Chromosomal Missegregation Phenotype. Close examination of the determined, a result of 4.7% for normal embryos (n ϭ 23) and Giemsa-stained, degenerating cultured Cenpa null embryos at 1.1% for Cenpa null embryos (n ϭ 14) was obtained. The days 5.5 and 6.5 revealed evidence of a severe chromosomal chromosomes seen in the null embryos appeared morphologi- missegregation phenotype. At day 5.5 (Fig. 3 e, g, and h), a cally more condensed and scattered than those of normal substantial number of micronuclei, formed from lagging chro- embryos, as was previously described for the Cenpc and Incenp mosomes, was observed. Some macronuclei indicative of en- null embryos (10, 21) (not shown). Except for an increased larged genomic content caused by failure of chromosomes to background of highly condensed chromatin bodies, no discern- divide properly were apparent. Chromosomal lagging or polar ible mitotic chromosomes were apparent in the 6.5-day null missegregation also affected normal cytokinesis, resulting in embryos examined, suggesting cessation of mitosis at this point. nuclear bridging and blebbing of the nuclear membrane. Chro- matin fragmentation and hypercondensation were apparent. Immunofluorescence Studies. Embryos were analyzed by using Similar mitotic problems were observed in the day-6.5 embryos antibodies raised against mouse Cenpa (36) or Cenpc (10) or an except for an increased degree of severity (Fig. 3 f, i, and j). At autoimmune serum (CREST#6) that recognizes human CENP-A and CENP-B (37). Fig. 4 shows typical results obtained on interphase cells in day-5.5 embryos. All three antisera gave Table 2. Correlation of genotype with morphology of cultured strong, discrete signals on the interphase cells of normal embryos embryos (Fig. 4 a–c). In the Ϫ͞Ϫ embryos, with the exception of the Time of Condition of No. of embryos occasional cell showing weak residual Cenpa staining, most Genotype harvest, days embryo (% total) interphases were negative for this antibody (Fig. 4d), suggesting a complete or near complete depletion of Cenpa (from maternal ϩ͞ϩ 8.5 Healthy 9 (26) cytoplasm) by this stage. The CREST#6 antiserum gave positive ϩ͞Ϫ 8.5 Healthy 19 (54) signals on the Ϫ͞Ϫ embryos (Fig. 4e), but the signals were Ϫ͞Ϫ 6.5 Degenerating 5 (15) discernibly different from those seen in the normal embryo in No PCR result 6.5 Degenerating 2 (5) that there was high background staining throughout the nucleus Total no. of embryos 35 (100) and the enhanced signals on chromatin were unusually diffuse Embryos were flushed from the uterus at day 3.5. Healthy embryos were through all focal planes. Because CENP-A was absent from these harvested at day 8.5 (i.e., 5 days in culture), while degenerating embryos were embryos, the observed CREST#6 signals could be attributed to harvested at day 6.5 (i.e., 3.5 days in culture) for nested PCR analysis. the staining of CENP-B. When tested with the anti-Cenpc

Howman et al. PNAS ͉ February 1, 2000 ͉ vol. 97 ͉ no. 3 ͉ 1151 Downloaded by guest on September 29, 2021 Fig. 4. Immunofluorescence analysis of day-5.5 embryos. (a–c) Wild-type interphase cells stained with anti-Cenpa, CREST#6 autoimmune serum, and anti-Cenpc, respectively. (d–f) Cenpa null interphase cells stained with anti-Cenpa, CREST#6, and anti-Cenpc, respectively. Although these pictures represented the results taken at one focal plane of a three-dimensional interphase cell nucleus, direct microscopic analysis through all the planes indicated only variation in the total number of observable signals but not the morphology of the signals (e.g., the more diffuse spots in e, and the higher background signals throughout the nuclei in both e and f compared with their respective controls). (Left) Simultaneous staining of chromatin with 4Ј,6-diamidino-2-phenylindole (blue) and centromere with anticentromere antibody (red). (Right) Split image of Left showing anticentromere antibody staining (red) only.

antibody, the prominent and discrete signals seen in normal impairment becomes apparent. Chromosomal missegregation embryos were undetected in the Ϫ͞Ϫ interphases (Fig. 4f). results in the formation of a large number of micronuclei Instead, these interphase cells showed a profuse speckling of (because of lagging chromosomes), macronuclei (because of Cenpc signals throughout the entire nucleus. nonseparated ), and nuclear bridging and blebbing (because of tethering chromosomes affecting cytokinesis). The Discussion detection of highly condensed, dark Giemsa-staining bodies Our gene targeting construct was designed to cause a premature reflects shrinking chromatin that prompts speculation of apo- translational termination and deletion of the centromere tar- ptotic cell death (38). geting domain of Cenpa. Evidence that complete gene knockout Immunofluorescence analysis has provided insight into the has been achieved comes from the complete absence of immuno- cause of mitotic disarray and the role of Cenpa. Depletion of fluorescence-detectable Cenpa proteins in morphologically de- Cenpa in day-5.5 Ϫ͞Ϫ embryos is accompanied by a significant generating Cenpa null embryos. The apparently normal pheno- alteration in Cenpb binding in the interphase cells. Instead of the type of the Cenpa-targeted heterozygous ES cell line and mice usual discrete and compact signals, Cenpb binding on interphase also indicates that our gene disruption strategy does not produce chromosomes now becomes more diffuse, suggesting that the any observable dominant-negative effect. local chromatin is no longer as highly condensed as that for a In previous studies, we reported that targeted gene disruption normal interphase centromere (compare Fig. 4 e with a–c). This of the centromere proteins Cenpc and Incenp in mice results in disrupted chromatin structure also appears to affect the normal preimplantation embryonic lethality before day 3.5 pc (10, 21). sequestration of Cenpb from the nuclear pool as evident from In comparison, expression of a severe phenotype in the Cenpa the increased Cenpb signal seen throughout the nucleus. At null embryos appears slightly delayed because these embryos are present, it is unclear what the role of Cenpb is because the able to implant into the uterus. Embryonic cell division and protein appears to be nonessential for normal mitotic and development up to this stage presumably are sustained by meiotic cell divisions (5–7). residual maternal cytoplasmic Cenpa protein (the presence of Anti-Cenpc antibody has been used to further investigate which has been demonstrated in our immunofluorescence ex- whether a structurally normal interphase kinetochore is formed periments on these early embryos; data not shown). The slightly in Cenpa-deficient cells. Cenpc is a good marker for this because longer survival time for Cenpa null embryos could reflect a the protein is present only on active centromeres (39, 40) and is greater stability of the maternal Cenpa mRNA and͞or its functionally essential (10). Our results indicate that depletion of translated protein compared with those of Cenpc and Incenp. As Cenpa leads to an absence of discrete Cenpc binding to inter- the maternal Cenpa becomes depleted at day 5.5 pc, mitotic phase centromere. As with Cenpb, sequestration of nuclear

1152 ͉ www.pnas.org Howman et al. Downloaded by guest on September 29, 2021 Cenpc also becomes impaired, resulting in a diffuse and speckled time of DNA replication, a chromosomal region becomes distribution of the noncentromere-targeted protein throughout marked for kinetochore assembly (29). A possible role of the nucleus. The profuse interphase staining seen with both replication not withstanding, the results of the present study anti-Cenpc and anti-Cenpb antibodies in the Ϫ͞Ϫ embryos also indicate that CENP-A is required for the kinetochore targeting indicates that these proteins are not limiting but are incapable, of functionally critical CENP-C and, by virtue of its nucleo- in the absence of Cenpa, to precipitate kinetochore formation. somal nature (expected to play a commanding role in estab- The observed failure of a structurally normal kinetochore to be lishing the primary level of chromatin packaging before the properly assembled during interphase provides a suitable expla- seeding of other kinetochore proteins such as CENP-C) nation for the progressive deterioration of mitotic chromosomal probably constitutes one of the earliest events in the interphase segregation in the Cenpa null embryos, leading ultimately to kinetochore assembly pathway. These properties are consis- severe mitotic disarray and embryonic cell death. tent with an epigenetic role of CENP-A in marking a chro- Recent data suggest that centromere formation does not mosomal region for centromere formation. Future studies strictly depend on DNA sequence and that epigenetic factors aimed at understanding the mechanisms that facilitate the may be involved (41–43). Such epigenetic factors are assumed recruitment of CENP-A to a potential centromeric site should to operate at the higher-order organizational level, directly on provide further important insight. chromatin structures. The histone H3-like property of CENP-A means that the protein can directly influence cen- We thank P. Mountford for the IRES-␤geo marker, G. Kay for the 129͞1 tromere-specific chromatin organization at the nucleosomal cell line, E. Robertson for STO͞NeoR feeder cells, S. Gazeas, M. Sibson, level, making this protein a suitable candidate for epigenetic A. Sylvain, and J. Ladhams for excellent technical assistance, and E. regulation. The observation that CENP-A synthesis appears to Earle for helpful discussion. This work was supported by the National be coupled with centromere replication (28) has prompted the Health and Medical Research Council of Australia. K.H.A.C. is a proposal that through direct incorporation of the protein at the Principal Research Fellow of the Council.

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